A broad panel of vaccine antigen candidates was evaluated for recombinant expression in both HEK cells (by UCPH) and Drosophila S2 cells (by Expres2ion). Early-stage efforts focused on the Nipah virus G and F proteins, as well as G proteins from related strains (Bangladesh, Malaysia) and other henipaviruses (Hendra, Ghana, Langya). In total, approximately 100 antigen constructs were generated, each genetically fused to either a Tag or Catcher domain to facilitate site-specific conjugation to a capsid Virus-Like Particle (cVLP). These constructs were tested across a range of cVLP-display strategies—including multivalent antigen designs—and within both protein- and mRNA-based vaccine platforms. Multiple linker configurations were also assessed to optimize expression, cVLP coupling efficiency, and antigen stability.
Recombinant expression in S2 cells yielded high levels of non-aggregated antigens that retained correct conformation, as confirmed by binding to conformation-sensitive monoclonal antibodies. These antigens could be efficiently coupled at high density to the AP205 cVLP, forming stable antigen:cVLP complexes. Selected lead candidates were subsequently evaluated for immunogenicity in mice, where they induced strong ELISA-binding and virus-neutralizing antibody responses.
In parallel, a genetic vaccine platform was used to identify antigen candidates suitable for either protein- or mRNA-based vaccine development. This approach supported the selection of the protein vaccine lead and served as a mitigation strategy by enabling identification of promising mRNA vaccine candidates. Antigens were initially screened for expression in HEK cells and evaluated for their ability to form secreted antigen:cVLP complexes. Promising designs were evaluated for immunogenicity in mice. The mRNA vaccines induced strong antigen-specific ELISA-binding titers as well as high virus-neutralizing responses.
Direct comparison of selected constructs delivered as mRNA or protein-based cVLP vaccines demonstrated that both vaccine platforms succeeded in inducing robust ELISA antibody titers, comparable to those measured in mice vaccinated with a control NiV vaccine, similarly to mice surviving a lethal dose of NiV challenge. These antibody levels also correlated with potent neutralization of Nipah virus, surpassing titers observed in convalescent human sera (WHO standard) and benchmark vaccine candidates.
Furthermore, cellular immune responses were evaluated by measuring antigen-specific CD8⁺ T cell responses in the spleens of immunized mice. Here, only the mRNA-based vaccines elicited robust CD8⁺ T cell responses, which were not observed following immunization with protein-based cVLP vaccines.
